Since the discovery of insulin by Banting and Best in 1921, the hormone, for human use, has been extracted from the pancreata of domestic animals, mainly cattle and pigs. While the supply of pancreas has been sufficient for industry needs to the present time, there has always been a desire for an alternative supply of the hormone. Thus, complete chemical synthesis has been achieved but is time-consuming and very expensive when compared to extraction of the natural tissue. The functional units of the pancreas that produce and mediate the production of insulin are the Islets of Langerhans. Although the Islets of Langerhans are numerous, they are widely dispersed throughout the pancreatic tissue and constitute only 5% of the total cells of the whole organ. Within each islet of the three different constituent cell types, alpha, beta and delta, it is known that only the beta cell produces insulin, while the alpha and delta cells produce glucagon and gastrin respectively.
In the present state of the art of animal cell culture, there is a central dogma that normal somatic cells from higher animals, when cultivated outside the body, progress through three phases (see L. Hayflick, Experimental Cell Research. 37: 614, (1965)). Phase I is an induction period during which the cells become adjusted and in which there is limited or no growth. Phase II is a period of exponential growth, where only a finite number of population doublings are possible and this doubling potential is characteristic of the species, i.e. approximately 50 doublings for man and for cattle approximately 12 doublings, etc. A normal, homogenous, cell population in Phase II is called a "cell strain" and a necessary condition for multiplication during this phase is attachment to a surface, when growth proceeds in two dimensions (monolayers). Because of the limited number of doublings of normal cells, most cell strains are started from foetal tissue, thus allowing the maximum number of population doublings before the cells enter Phase III which is near the ultimate population doubling and when the cells become moribund and soon die. Occasionally in late Phase II, a genotypic and phenotypic alteration occurs when new cells appear that grow in fluid suspension and replicate indefinitely. Such a population, when homogenous, is termed a "cell line".
The present invention provides a novel continuous cell line that is morphologically very similar to the beta cell of the Islets of Langerhans and which is capable of producing insulin. The continuous cell line is obtained by transformation of bovine pancreas cells and has the unusual feature that they contain the diploid number of chromosomes for the bovine species (2n=60). Most transformed cells contain either more or fewer chromosomes than the normal cell.
A continuous cell line in accordance with this invention has been designated CLL 36A/76 and is deposited with American Type Culture Collection of Rockville, Md., under accession number ATCC CRL 1407. Although this indicated public availability is the simplest method of obtaining a cell line in accordance with this invention, it is not altogether impossible and improbable that similar and functionally identical bovine beta cell lines may be produced by procedures other than those described herein. Such functionally and morphologically substantially identical cell lines are equivalent to cell line 36A/76 and are included within the scope of this invention.
The continuous cell line is generally provided as a cell culture of the cell line in a nutrient culture medium. The nutrient culture medium preferably is that known by the trademark CMRL-1969.
CMRL-1969 is a well known nutrient culture medium whose properties and composition are described in detail in G. M. Healy et al., Applied Microbiology, vol. 21, 1 (1971). Although this material is identified by trademark, this is sufficient description for a person skilled in this art to identify the nutrient medium adopted.
The CMRL-1969 may be supplemented in the cell culture by calf serum, preferably foetal calf serum.
The insulin which is produced by the cell line may be extracted from centrifuged cells of the cell line by conventional biochemical extraction, for example, using acidic alcohol solvent. The insulin is readily recovered in solid form from the solvent.
The continuous cell line is produced from bovine pancreas tissue. The majority of the exocrine tissue is first removed from pancreatic tissue obtained from a viable calf embryo. The cells are transferred to a specific growth medium and allowed to become confluent.
These primary tissues are judged by observation and production of insulin as to whether they are essentially homogenous or are a mixture of cell types. If homogenous, the cells are propagated by standard methods in tissue culture flasks, or occasionally, if large numbers of cells are desired, in a multi-surface cell propagator(MSCP). Usually, shortly after the 12th passage the cells enter Phase III and die. Occasionally, in about 3% of such attempts, cells in Phase III do not die but undergo transformation into the cell line of this invention.
While the transformation of the Phase III cells into the cell line occurs in only about 3% of the cultures, the transformation nevertheless does occur in a repetitive manner. The transformation of Phase III cells into a continuous cell line according to this invention, is reproducible, although perhaps somewhat tedious to effect. The continuous cell line of this invention, therefore, results from a transformation of Phase III cells which occurs in a low percentage of attempts, rather than from a single chance transformation as is commonly the case with cell line formation.
Throughout Phase II of the propagation of the cells, application of known secretagogues, such as glucagon, to the cell cultures will stimulate the release of insulin into the medium, and the insulin may be separated by selective membrane (AMICON) filtration well-known to the art. After the cell line has formed, it no longer secretes insulin into the medium.